PROJECT SUMMARY Despite decades of research, Staphylococcus aureus (S. aureus) remains a major threat in neonatal intensive care units (NICUs), as the second leading cause of bloodstream infection, ventilator-associated pneumonia, and surgical site infections. Rates of invasive S. aureus infection in neonates far exceed rates of infection in older children and adults. Novel strategies are needed to prevent S. aureus disease in this population. Some neonates that are exposed to S. aureus become colonized, whereas others do not become colonized. Prior, mostly cross-sectional, studies have suggested that cultured commensal skin bacteria, including coagulase- negative Staphylococci and Corynebacterium spp. may compete with and exclude S. aureus from the nasal microbiota. 16S rRNA gene amplification and sequencing techniques can provide a better understanding of the nasal microbiota, including the interactions and functional characteristics of bacterial communities, to inform new strategies for the prevention of neonatal S. aureus colonization and disease. For example, a single species may not be sufficient to resist S. aureus colonization, but a community of organisms may exist that protects against colonization. Our long-term goal is to further understand the nasal microbiota and develop novel interventions for prevention of neonatal S. aureus infections. We propose to characterize the dynamic nasal microbiota of neonates in the NICU using 16S rRNA gene amplification and sequencing techniques using stored specimens from a federally-funded randomized controlled trial. The Specific Aim is to characterize the dynamic nasal microbiota in our existing cohort of hospitalized neonates at risk for S. aureus colonization and to identify bacterial communities that may provide resistance to S. aureus colonization, and the Sub Aim is to identify clinical characteristics, such as antibiotic exposure and parental S. aureus colonization status, which may impact the dynamic nasal microbiota and reduce resistance to S. aureus colonization. A matched case- control study will be conducted using clinical data and stored weekly nasal swabs from 50 hospitalized neonates who acquired S. aureus and 100 hospitalized neonates who did not acquire S. aureus colonization. By combining 16S rRNA gene amplification and sequencing techniques with our unique patient cohort and stored samples, we will characterize the nasal microbiota and offer insights into how bacterial communities interact to promote colonization resistance against S. aureus. This proposal builds on our previous work to develop novel strategies to prevent S. aureus disease in vulnerable neonates. The proposed work will lay the foundation for targeted microbiome studies as a potential preventive strategy in a high-risk, minority, population.